Photonic crystal enhanced fluorescence emission and blinking suppression for single quantum dot digital resolution biosensing

While nanoscale quantum emitters are effective tags for measuring biomolecular interactions, their utilities for applications that demand single-unit observations are limited by the requirements for large numerical aperture (NA) objectives, fluorescence intermittency, and poor photon collection efficiency resulted from omnidirectional emission. Here, we report a nearly 3000-fold signal enhancement achieved through multiplicative effects of enhanced excitation, highly directional extraction, quantum efficiency improvement, and blinking suppression through a photonic crystal (PC) surface. The approach achieves single quantum dot (QD) sensitivity with high signal-to-noise ratio, even when using a low-NA lens and an inexpensive optical setup. The blinking suppression capability of the PC improves the QDs on-time from 15% to 85% ameliorating signal intermittency. We developed an assay for cancer-associated miRNA biomarkers with single-molecule resolution, single-base mutation selectivity, and 10-attomolar detection limit. Additionally, we observed differential surface motion trajectories of QDs when their surface attachment stringency is altered by changing a single base in a cancer-specific miRNA sequence.

The manuscript by Xiong et al. reports a thorough and impressive study of the use of enhanced quantum dot emission from a grating surface for implementing a digital miRNA assay. The improvements afforded by the optical enhancements are cleverly leveraged to make significant improvements to conventional microscope assays. In particular, the fact that only the quantum dots that bind to the miRNA target as part of the assay sandwich become bright, is a very elegant way to deal with potential background signal from unbound dots. The descriptions and figures are very detailed and comprehensive and support the narrative very well. The manuscript is suitable for publication in Nature Communications if the authors can address the following issues: -The analysis of the optical improvement in the presence of the photonic crystal grating is very clear and well described. However, the effect seems to be essentially identical to what the group reported in a seminal paper in Nature Nanotechnology over a decade ago. It would be helpful if the authors could clarify if and how the present demonstration differs from their own work (refs. 27-29).
-The assay appears to be a sandwich assay. It may be simpler to just call it that. -The authors did carry out simulations for a negative control experiment with non-matching miRNAs. However, the manuscript would have been significantly strengthened if a negative control experiment were reported and if the miRNAs were detected from a more meaningful (native) sample matrix. Were any of these carried out? -While it may be correct that TIRF microscopy has a dynamic range of 5 logs, there have been numerous reports of lab-on-chip based digital assays with significant larger dynamic range. Providing better context for the present work might be helpful. - Fig. 4b reports an impressive performance of the sensor 9 orders of magnitude in concentration. However, the number of detected quantum dots only increases by about a factor of 10, suggesting an extremely nonlinear performance with very little wiggle room for distinguishing vastly different concentrations. Shouldn't the assay be linear? The authors should provide a discussion of this nonlinearity. Can the observed relationship between number of targets and detected number of quantum dots be modeled? -The sensor has a relatively long incubation time of 2 hours. How was that chosen? Can it be realistically improved? Can the authors add a figure that shows the performance of the sensor (# of detected QDs) versus incubation time? -Related to the previous issue, how effectively are the QD probes and target molecules transported to the sensor surface? Can the authors provide more details on how the complete sensor looks like in terms of amount of fluid probed, dimensions of the compartment etc.? An image of the complete device (perhaps in the supplemental information) would help understand the full system arrangement.
Reviewer #2 (Remarks to the Author): In this manuscript, the authors demonstrated that the photonic crystal can greatly enhanced the excitation, directional extraction, and blinking suppression for single quantum dot digital resolution biosensing. It is an attempt to gain more control over photonic crystal enhancement of quantum dot luminescence and is thus of interest to the broad research community of nanophotonics and biosensing. The paper is recommended for publication after addressing the comments below.
1. On page 9, the authors state that high concentration drop-casted QDs (1uM) spread uniformly on the PC surface without aggregation (Figure 1a). Does this mean a monolayer of QD was formed on PC surface? A SEM image of a small area (500 nm x 300 nm) is not sufficient to confirm the uniformity. A statistical analysis of multiple images will be helpful to confirm this. There is a concern that the density of QDs is heterogeneous due to the coffee ring effect associated with drop-casting preparation. 2. What is the size uniformity of QDs? What is the distance between QDs and the PC surface? How does the distance/size uniformity of QDs affect the enhancement? 1. Firstly, there is a general question as to the value of this technology, analytically. With sample preparation taking several hours and measurement then taking an addition 2 hours, how valuable is this to clinical practice. The authors should more clearly articulate a vision as to why this is important to the wider readership of Nature Comm (put another way, what is the clinical decision and care pathway that will be critically affected by whether there is 10aM or 100aM of miRNA in a sample ?) 2. I would also argue that the introduction is not written to identify novelty. There are a lot of references cited for every part of the system, so the authors are indicating that the only novelty is in the integration. The authors have to clearly show that this is more than simply the sum of the parts, which is not at all clear in the writing in the current form.
3. On the subject of (2), the claim of a X3000 fold enhancement is relative to a glass slide as reference measurement. What is more interesting to me as a reviewer and indeed to the readership is what the improvement is, relative to other nanostructured photonic systems, expecially those using photonic crystals. Again, a detailed critical view is needed.
4. The paper would also greatly benefit from re-organisation of the figures. The multiple panels serve only to confuse the reader -there is a strong case for some of these to be moved to ESI (especially in Fig 1., as the panels do not evidence the major point of the paper). I think the authors need to make decisions on key data that corroborates the message(s) in the paper. I believe the paper would be more understandable if Figure 3 was actually Figure 1 and was referenced in the introduction.

Specific Details
Most of my specific comments are focussed around Figure 4 -which I believe is the key result. As advice, by combining both static and dynamic measurements into a single panel this obscures and confuses -this data should sit within 2 separate figures, associated with the two sections in the text. This is the key experimental evidence and readers must be allowed to understand it.
1. Figure 4a has different scaling in inset is also difficult to interpret. Both images at the same scale should at least be provided in Supplementary Information, with the same resolution. Rather than see all 9 concentrations (+ background) better to show less at the same resolution to allow for a critical comparison.
2. Figure 4b is too difficult to understand with the double axis with different scales. I suspect the reference should also be the background unmodified glass (grey), not the noise (blank +3sd, blue), so that the authors show how much better their device performs relative to an unstructured glass slide. The signal to background would be 3 sd above the grey, not blue, line.
3 . Fig 4 b, Why did the experiment stop at 10 aM? Given the linear plot over 9 orders of magnitude concentration, and that the signal should still be apparent above the background at 2 orders of magnitude below the current LOD, at 100zM, why did the authors not extend this investigation. If the linear relationship does indeed extend at lower concentrations against the background, this should be shown. Similarly if it does not, it should still be shown so as to give a better understanding of the system.
The same is true at the high concentrations to provide a complete understanding of the technique. Why are measurement above and below the linear range not shown. As a final comments, some thought about significant figures and errors -e.g 1.708ns (+/-0.3nS). Does this measurement really have a precision around 1 ps or is this algorithmic from the fitting function used in Fig 2(g), and what does that value mean when the error is 2 orders of magnitude higher than the measurement. Do the authors mean 1.7 ns (+/-0.3nS) which is less convincing, or is this a typographic error e.g. 1.708ns (+/-0.3pS). Figure 4(i), the reader should have a view of variability (how do the 5 trajectory measurements compare)? Another example of important information almost lost by the compression of 9 figures into one panel, and by merging the static and dynamic data in the same figure.